Albumin detecting method and means



United States Patent N0 Drawing. Application -May 8, '1957 Serial No. 657,731.

Claims. (Cl. .23 230)..

This invention-relates to animproved method and means for the detection of proteinaceous materials. More particularly, this invention relates to an improved method and means for the detection of protein in bio logical fluids. In one specific embodiment thereof, this invention relates to an improved method and means for the detection of albumin in urine.

The presence in urine of more than traces of albumin is almost invariably an indication of 'a pathological condition and accordingly the detection of albumin in urine is a testing procedure of great clinical importance. Albuminuria is likely to be observed in arteriosclerosis, hypertension, nephritis, diabetes, kidney stones, rheumatic fever, certain contagious fevers, many cardiac conditions, and in several other serious ailments.

At the present time the methods used for the detection of albumin in urine are all based on the formation of a turbidity or precipitate resulting from the coagulation of the albumin present by a suitable chemical reagent (usually a strong acid) or by a combination of a suitable chemical reagent and heat. The only significant difference between the many tests presently used in clinical practice resides in the nature of'the reagent employed to coagulate the albumin. Sulfosalicylic acid, picric acid, nitric acid, sodium hexametaphosphate, acetic acid plus heat, and the like are in common use. To be reliable, these turbidity or precipitation tests must be performed on urine specimens that have been carefully clarified, since turbidities or precipitates caused by substances suchas pus, bacteria, and the like, can easily be mistaken for'albumin. The urine sample is preferably centrifuged before testing in order to remove any such interfering materials or, more frequently, the sample is merely filtered to achieve clarification although in many instances simple filtration is not sufiicient to remove such interfering substances.

The disadvantages of present methods for the detection of albumin in urine are quite evident. Such methods require special equpment which is frequently unavailable at the physicians ofiice and more especially at the bedside of the patient (such as a centrifuge or filtration means, a source of heat, and the like), require the use of corrosive acids and are based on the reading of a turbidity or' the detection of a precipitate which often requires a trained eye for reliable results.

Considerable effort has been devoted to an attempt to develop, a. colorimetric method for the detection of albumin'in urine. These eiforts have usually sought to take advantage of the protein error exhibited by certain indicators; As is well known, acid-base indicators undergo an abrupt and'decided. change in color at a certain pH value, the exact .pH value at which the change occurs being determined by the nature of the particular indicator concerned However, certain acid-base indicators, .when present in liquids containing proteinaceous materials, do not exhibit their characteristic color change at'thesame pH value that "brings about this color change in the absence of proteinaceous materials. It is postulated 2,897,058 Patented July '28,. 1959 "ice that the indicator reacts with the proteinto form a com pound similar to a salt, this material being more stable than the usual alkali metal or similar salts of the indicator. In the absence of proteinaceous materials the alkali metal or similar salt of the indicator, which-has a characteristic color, is converted to. the free indicator (of a decidedly different color than the salt form of the indicator) at a certain pH. However, in the presence of proteinaceous materials, this pH is not adequate to decompose the protein-indicator compound or complex. A still lower pH value is necessary to decompose the indicator-protein compound or complex and thus produce the characteristic color of the free indicator. I

Esters (for example, the ethyl ester) of tetrabromophenolphthalein (usually employed in the form of an alkali metal, particularly the potassium, salt) exhibit a very, pronounced protein error. The alkali metal salts of this indicator are blue While thecolor of the free indicator is yellow. The protein compound or complex of this indicator is also blue and is much more stable than the alkali metalvsaltsthereof. While dilute acetic acid'converts the blue alkali metal salts of this indicator to the yellow free indicator, the blue indicator-protein compound or complex remains unchanged in the presence of dilute acetic acid and can only be decomposed to the yellow free indicator by addition ofconcentrated acetic acid or a mineral acid.

From theabove discussionit is evident thatthe protein error phenomenon presents at least the possibility of developing a colorimetric method for the detection ofalbumin in urine. On adding an alkali metal salt ofan indicator exhibiting a pronounced protein error efiect' to urine containing albumin, the indicator-protein compound or complex forms. Now if acid is added tothe result ing mixture of a kind and in an amountsufiicient to give a pH value between thatnecessary to decompose the alkali metal salt of the indicator and that necessary to decompose the indicator-protein compound or complex, the characteristic salt form. color of the indicator will persist unchanged due to the presence of undecomposed indicator-protein compound or complex. It, on the other hand, an alkali metal salt of the indicator is added to a sample of albumin free urine and the resulting mixture is: brought to the previously mentioned pH value intermediate of that necessary to decompose the alkali metal salt of the indicator andthat necessary to decompose thecompound or complex the indicator forms with proteins, the characteristic color of the salt form of'the indicator will be completely discharged and replaced by the characteristic color of the free indicator.

It has been proposed to use two reagents in such a test procedure, one a solution of a tetnabromophenolph thalein ester in methanol or ethanol, the other a solution of an acid, such as acetic acid, in'water. The urine is first treated with the solution of theindicator, whereupon it turns blue due to the presence of mildly alkaline materials such as phosphates, carbonates, urates, and the like, and due to albumin, if present. Then this blue solution is treated with the solution of the acid. If the blue color disappears, the'urine is free of albumin; if the bluecolor persists, then albumin is present.

Obviously, this procedure is far from ideal. Two separate reagents must be handled and used in certain relative proportions which are critical. If an insufficient amount of acid is added to the blue solution, the blue color of the salt form of the indicator will not be discharged and the reading will be positive even though albumin is absent. Conversely, if an excessive amount of acid is added inadvertently, .the blue color may be discharged even though albumin is present and a false negative test will be the result.-

A further and even more fundamental difficulty with the proposed procedure resides in the fact that the end point is not sharp. At low but still definitely pathological albumin contents (about 0.015% to 0.02% albumin on urine) the color produced on. bringing the sampleindicator mixture to the proper pH is somewhat green and is difficult to distinguish from the yellowish color of the free indicator. While urines containing appreciably more than 0.02% albumin produce an unmistakable blue color in this test it is diflicult or impossible to distinguish between an albumin free urine and one containing in the neighborhood of0.02% albumin or less by this test.

Attempts have been made to simplify this test procedure, as far as manipulative details are concerned, by combining the alcoholic solution of the ester of tetrabromophenolphthalein and the acetic acid into a single solution. However, the resulting combined reagent decomposes within a week or two, giving a colorless solution that is completely useless for the detection of albumin. Also, such a combined reagent exhibits the previously mentioned uncertainties when applied to urine samples containing in the neighborhood or 0.02% albumin or less.

As a result of these difficulties and uncertainties, the detection of albumin by the color reaction witha tetrabromophenolphthalein ester has been adjudged unreliable and is not employed in clinical practice.

I have discovered a new composition of matter which combines an ester of tetrabromophenolphthalein and an acid in a single solution which is stable over protracted periods of time and constitutes a test solution for the reliable colorimetric detection of pathological quantities of albumin in urine.

One object of my invention is to provide a stable, single test solution for the reliable detection of albumin in urine.

An additional object of my invention is to provide a reliable method for the colorimetric detection of albumin in urine that requires a minimum of equipment and manipulation.

Additional objects of my invention will become apparent as the description thereof proceeds.

I have discovered that a solution containing an ester of tetrabromophenolphthalein plus an acid in the concentrations suitable for the colorimetric detection of albumin in urine is stable over protracted periods of time providing that the solution is made using certain tertiary alcohols (that is, alcohols in which the characteristic OH group is attached to a carbon atom which, in turn, is attached to three other carbon atoms) as the solvent. While there are many tertiary alcohols, in view of the conditions that obtain in the test procedure of this invention only a very few of these tertiary alcohols are suitable for use as solvents in preparing the unitary test solution of this invention. Urine is predominately aqueous and since the mixture of urine and test solution must be homogeneous only water soluble tertiary alcohols are suitable for use in this invention. Tertiary alcohols must contain at least four carbon atoms and, as is well known, the dividing line between complete miscibility and limited solubility or complete insolubility in wateraliphatic alcohol systems occurs when the number of carbon atoms in the alcohol is four or more. Thus, while t-butyl alcohol is infinitely soluble in water, t-amyl alcohol is only slightly soluble in water. There are only two common alcohols that satisfy the two criteria of being tertiary alcohols and water soluble alcohols. These are t-butyl alcohol and diacetone alcohol.

I have discovered that a unitary test solution prepared using one of these two tertiary alcohols specifically mentioned above or a mixture of these two alcohols as the solvent not only is stable over protracted periods of time but also sharply differentiates between albumin free urines and urines containing a small but pathologically significant quantity of albumin.

It was totally new and unexpected to find that acid solutions of a tetrabromophenolphthalein ester in t-butyl 4 alcohol and/or diacetone alcohol are stable for periods of a year or more, in view of the known fact that these solutes in such solvents as methanol, ethanol, propanol, i-propanol, glycols, dioxane, and the like are unstable and decompose within a short time, usually within two or three weeks.

Also, it was totally new and unexpected to find that acid solutions of a tetrabromophenolphthalein ester in t-butyl alcohol and/ or diacetone alcohol difierentiate with certainty between albumin free urines and urines containing a small but pathologically significant amount of albumin, in view of the known fact that this differentiation is very uncertain and questionable when the test solution consists of these solutes dissolved in methanol, ethanol, propanol, i-propanol, glycols, dioxane, and the like.

Example 1 This example demonstrates the instability of test solutions prepared using methanol and solvents of a similar nature.

One tenth gram (0.1 g.) of tetrabromophenolphthalein ethyl ester was dissolved in ml. methanol and 8 m1.- acetic acid were added. Two drops of the freshly prepared solution added to five drops urine containing albumin produced a blue color. With albumin free urine the color obtained is green or yellow.

After standing for two weeks this reagent solution, which was deeply colored when fresh, became light in color. When employed as above described to test urine containing the same amount of albumin as the sample employed previously, only a weak, indistinct blue color was produced.

After standing an additional two weeks (four weeks total) the reagent solution became almost colorless and gave no reliable indications with a urine specimen containing the same amount of albumin as the sample of albumin containing urine employed in connecion with the freshly prepared solution.

When ethyl alcohol, propyl alcohol, i-propyl alcohol; glycol, dioxane, was substituted for the methanol solvent in the above formulation the results were essentially the same, the reagent decomposing and becoming useless as a test solution in a matter of two or three weeks, with some of these solvents in twelve days or less.

Example 2 One tenth gram (0.1 g.) of tetrabromophenolphthalein ethyl ester was dissolved in 100 ml. t-butanol and 8 ml. acetic acid were added. Two drops of this solution added to urine (5 drops) containing the same amount of albumin as the albumin containing urines of Example 1, produced a blue color. With urine free of albumin the color produced is green or yellow.

This reagent was employed as a test solution in the same manner atfrequent intervals. After 14 months it was still fully reliable as a test for albumin, giving a distinct blue color when albumin was present and a green or yellow color in the absence of albumin.

When diacetone alcohol was substituted for the t-butyl alcohol of this formulation a reagent for albumin of similarly high stability was obtained, which was still fully reliable after more than one year of testing.

Example 3 The reagent prepared in accordance with Example 2 has been applied to a large number of urine specimens in various hospitals and clinics and the results obtained checked against those obtained using established tests such as the sulfosalicylic acid and the heat plus acetic acid turbidity tests, the nitric acid ring test (Hellers test) and the like.

Agreement between the results obtained by use of the colorimetric test of this invention and the results obtained in parallel tests employing one or more of the conven-v tional procedures mentioned was observed with 97% of the specimens. A retesting of the specimens involved in the comparatively few disagreements resolved the matter in favor of the-test solution of this invention, itbeing demonstrated that the turbidities observed in the original conventional tests were due to substances other than albumin. In this Work it was found that agreement among the various turbidity and precipitation tests was not complete and in borderline cases the decision whether the test was positive or negative depended in large measure on the experience and visual acuteness of the observer. However, as can be seen, the agreement between the conventional test results and those obtained with the test reagent of the present invention was quite good and it was concluded that the test procedure of the present invention is more reliable than the conventional test procedures previously mentioned, especially in borderline cases where the urine contains a small but pathologically significant amount of albumin.

Also, it is to be noted that the test procedure of the present invention requires only a dropper, a bottle of test solution (preferably provided with a dropping closure, e.g. an indicator bottle) 'and a spot plate.- Conventional test procedures require a centrifuge or filtering means, test tubes, frequently a source of heat, and the like. Also, conventional testing procedures require the use of strong and corrosive acids.

While this invention has been described largely in connection with the use of the ethyl ester of tetrabromophthalein, any available ester (methyl, propyl, et cetera) of this compound may be employed. Also, in place of using the free ester in the preparation of the reagent of this invention, a salt, for example, the potassium salt, of the selected ester may be used.

As has been explained previously, the purpose of the acid is to bring the urine-reagent mixture to a pH intermediate between that required to give the indicator in free (i.e. non-salt) form and that required to decompose the indicator-protein compound or complex. Neutral or alkaline urines give a blue color (i.e. the salt form) with tetrabromophenolphthalein esters regardless of whether albumin is present or not. On the somewhat acid side, on the other hand, the blue color is produced only in the presence of albumin. Acetic acid is generally used in clinical practice for the purpose of acidifying urines and other biological fluids because of its ready availability, convenient strength and solubility in most biological fluids. However, any other acid of similar properties and strength (dissociation constant about l.75 is equally suitable in the reagent of this invention, for example, propionic or butyric acid. Strong acids, such as chloroacetic, hydrochloric or sulfuric acids, are unsuitable since the blue compound formed by the reaction between tetrabromophenolphthalein esters and albumin is not stable in the presence of strong acids.

While this invention has been described almost exclusively in connection with the detection of albumin in urine it is obviously of much wider applicability. The test procedure of this invention may be employed to detect the presence of proteinaceous materials in biological fluids in general, for example, for the detection of globulin in spinal fluid, and even more broadly to detect the presence of proteinaceous materials in general in aqueous media.

Be it remembered that while this invention has been described in connection with specific details and specific embodiments thereof, these are illustrative only and are not to be considered limitations on the spirit or scope of 6 said invention except in so far as these may be incorporated in the appended claims.

I claim:

1. The composition of matter comprising a solution of a weak organic acid and an ester of-tetrabromophenolphthalein in a water soluble tertiary alcohol selected from the group consisting of tertiary butyl alcohol and diacetone alcohol. I

2. The composition of matter comprising a solution of acetic acid and the ethyl ester of tetrabromophenolphthalein in a water soluble tertiary alcohol selected from the group consisting of tertiary butyl alcohol and diacetone alcohol.

3. The composition of matter comprising a solution of acetic acid and the ethyl ester of tetrabromophenolphthalein in tertiary butyl alcohol.

4. The composition of matter comprising a solution of acetic acid and the ethyl ester of tetrabronrophenolphthalein in diacetone alcohol.

5. The process for detecting the presence of proteinaceous materials in an aqueous medium which comprises admixing with said aqueous medium a solution of a weak organic acid and an ester of tetrabromophenolphthalein in a water soluble tertiary alcohol selected from the group consisting of tertiary butyl alcohol and diacetone alcohol and observing the color of the resulting mixture.

6. The process for detecting the presence of proteinaceous materials in biological fluids which comprises admixing with said biological fluid a solution of a weak organic acid and an ester of tetrabromophenolphthalein in a water soluble tertiary alcohol selected from the group consisting of tertiary butyl alcohol and diacetone alcohol and observing the color of the resulting mixture.

7. The process for detecting the presence of globulin in spinal fluid which comprises admixing with said spinal fluid a solution of a weak organic acid and an ester of tetrabromophenolphthalein in a water soluble tertiary alcohol selected from the group consisting of tertiary butyl alcohol and diacetone alcohol and observing the color of the resulting mixture.

8. The process for detecting the presence of albumin in urine which comprises admixing with said urine at solution of a weak organic acid and an ester of tetrabromophenolphthalein in a water soluble tertiary alcohol selected from the group consisting of tertiary butyl alcohol and diacetone alcohol and observing the color of the resulting mixture.

9. The process for detecting the presence of albumin in urine which comprises admixing with said urine a solution of acetic acid and the ethyl ester of tetrabromophenolphthalein in tertiary butyl alcohol and observing the color of the resulting mixture.

10. The process for detecting the presence of albumin in urine which comprises admixing with said urine a solution of acetic acid and the ethyl ester of tetrabromophenolphthalein in diacetone alcohol and observing the color of the resulting mixture.

References Cited in the file of this patent Feigl: Mikrochimica Acta, volume H, 1937, pages 107-110.

Cohn: Indicators and Test Papers," page 165, 1899, John Wiley & Sons.

Allport: Colorimetric Analysis, p. 263, 1947, Chapman & Hall.

Jacobs: Handbook of Solvents, p. 160, 1953, D. Van Nostrand. 

1. THE COMPOSITION OF MATTER COMPRISING A SOLUTION OF A WEAK ORGANIC ACID AND AN ESTER OF TETRABROMOPHENOLPHTHALEIN IN A WATER SOLUBLE TERTIARY ALCOHOL SELECTED FROM THE GROUP CONSISTING OF TERTIARY BUTYL ALCOHOL AND DIACETONE ALCOHOL. 